Some molecular studies we had been doing for the identification of rapidly growing mycobacteria led us to examine more carefully the parameters involved in the carbon source utilization test for the identification of these organisms. This is a phenotypic test that may be very useful for distinguishing among certain species of rapid growers, either in those institutions that do not have the capability for molecular analysis or in those circumstances in which further study of an isolate is warranted. The optimal conditions for performing the carbon source utilization test, as well as its true discriminating power, have not yet been well defined.